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Am J Physiol Heart Circ Physiol 294: H2371-H2381, 2008. First published March 28, 2008; doi:10.1152/ajpheart.01279.2007
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Voltage-dependent K+ channels regulate the duration of reactive hyperemia in the canine coronary circulation

Gregory M. Dick,1 Ian N. Bratz,2 Léna Borbouse,2 Gregory A. Payne,2 Ü. Deniz Dincer,2 Jarrod D. Knudson,3 Paul A. Rogers,4 and Johnathan D. Tune2

1Department of Exercise Physiology, Center for Interdisciplinary Research in Cardiovascular Sciences, West Virginia University School of Medicine, Morgantown, West Virginia; 2Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana; 3Department of Pediatrics, Baylor College of Medicine, Houston, Texas; and 4Department of Physiology, Louisiana State University Health Sciences Center, New Orleans, Louisiana

Submitted 1 November 2007 ; accepted in final form 27 March 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We previously demonstrated a role for voltage-dependent K+ (KV) channels in coronary vasodilation elicited by myocardial metabolism and exogenous H2O2, as responses were attenuated by the KV channel blocker 4-aminopyridine (4-AP). Here we tested the hypothesis that KV channels participate in coronary reactive hyperemia and examined the role of KV channels in responses to nitric oxide (NO) and adenosine, two putative mediators. Reactive hyperemia (30-s occlusion) was measured in open-chest dogs before and during 4-AP treatment [intracoronary (ic), plasma concentration 0.3 mM]. 4-AP reduced baseline flow 34 ± 5% and inhibited hyperemic volume 32 ± 5%. Administration of 8-phenyltheophylline (8-PT; 0.3 mM ic or 5 mg/kg iv) or NG-nitro-L-arginine methyl ester (L-NAME; 1 mg/min ic) inhibited early and late portions of hyperemic flow, supporting roles for adenosine and NO. 4-AP further inhibited hyperemia in the presence of 8-PT or L-NAME. Adenosine-induced blood flow responses were attenuated by 4-AP (52 ± 6% block at 9 µg/min). Dilation of arterioles to adenosine was attenuated by 0.3 mM 4-AP and 1 µM correolide, a selective KV1 antagonist (76 ± 7% and 47 ± 2% block, respectively, at 1 µM). Dilation in response to sodium nitroprusside, an NO donor, was attenuated by 4-AP in vivo (41 ± 6% block at 10 µg/min) and by correolide in vitro (29 ± 4% block at 1 µM). KV current in smooth muscle cells was inhibited by 4-AP (IC50 1.1 ± 0.1 mM) and virtually eliminated by correolide. Expression of mRNA for KV1 family members was detected in coronary arteries. Our data indicate that KV channels play an important role in regulating resting coronary blood flow, determining duration of reactive hyperemia, and mediating adenosine- and NO-induced vasodilation.

ischemic vasodilation; adenosine; 4-aminopyridine; delayed rectifier potassium channel; vascular smooth muscle

IN THE CORONARY CIRCULATION, a brief period of ischemia is normally followed by a large and transient compensatory increase in blood flow. This phenomenon of reactive hyperemia, different from active (also known as functional or metabolic) hyperemia, is thought to represent a repayment of blood flow debt and is attributed to the accumulation of ischemic vasodilator metabolites. Evidence supports both adenosine and nitric oxide (NO) as mediators of reactive hyperemia (2, 4, 12, 52). Importantly, however, neither block of adenosine nor NO signaling can completely abolish reactive hyperemia (56). Thus the mechanisms of reactive hyperemia remain incompletely understood. Moreover, other mediators have been suggested, and it is likely that future studies will identify additional candidates. Rather than focus on putative metabolites underlying reactive hyperemia, we have turned our attention to possible end-effectors in vascular smooth muscle. K+ channels are likely targets of vasodilator metabolites, because K+ channels determine membrane potential and thus vascular tone (27, 35). Previous studies have focused on Ca2+/voltage-sensitive (BKCa) and ATP-dependent (KATP) K+ channels. To date, only one study suggests a role for BKCa channels (36) and published studies on KATP channels are conflicting; voltage-dependent (KV) K+ channels have not been investigated.

KV channels are possible end-effectors in reactive hyperemia. We have demonstrated (43, 44, 47) that KV channels participate in coronary vasodilation in response to H2O2 and increases in myocardial metabolism. The goal of the present study was to test the hypothesis that KV channels participate in coronary reactive hyperemia. As yet, there have been no in vivo studies, in humans or animals, that have aimed to determine the role of KV channels in regulating coronary reactive hyperemia; however, other K+ channels have been investigated as end-effectors of ischemic vasodilation. For example, Node et al. (36) demonstrated that bradykinin-induced activation of BKCa channels regulates blood flow in ischemic myocardium of dogs treated with NG-nitro-L-arginine methyl ester (L-NAME). When coronary blood flow was reduced to one-third of its baseline value, intracoronary (ic) administration of iberiotoxin, a selective BKCa channel antagonist, further decreased coronary blood flow. No studies in humans have addressed whether BKCa channels modulate coronary hemodynamics.

With regard to KATP channels in reactive hyperemia, some studies indicate a role, whereas others do not. For example, Banitt et al. (5) demonstrated that tolbutamide (a KATP channel antagonist), while without effect on resting flow or peak hyperemia, attenuated total hyperemic volume in the human forearm. In contrast, Farouque and Meredith (17) found that another KATP channel antagonist, glibenclamide, did not alter resting flow, peak hyperemia, or total hyperemic volume in the human forearm. To the best of our knowledge, the effect of KATP channel blockers on reactive hyperemia in the human coronary circulation has not been assessed. However, studies in humans with glibenclamide indicate that KATP channels regulate resting coronary blood flow, contribute to adenosine-induced coronary vasodilation, and participate in coronary metabolic vasodilation (18, 19). In the canine coronary circulation, KATP channels appear to mediate a portion of reactive hyperemia, as glibenclamide attenuates the peak and duration of reactive hyperemia (3). Subsequent studies in dogs and pigs have added support to a role for KATP channels in reactive hyperemia (9, 14, 28, 54, 58). A caveat for interpreting these studies is that high concentrations of glibenclamide may also block some KV channels (57). Since the role of KV channels in coronary reactive hyperemia is not known, we performed the present study.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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Animal care and use. An Institutional Animal Care and Use Committee approved our protocols, which were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, revised 1996). Male mongrel dogs (n = 36) were given morphine (3 mg/kg sc) as a sedative/preanesthetic and {alpha}-chloralose (100 mg/kg iv) as an anesthetic before surgical instrumentation. At the end of open-chest experiments, the heart was fibrillated. These methods of death conform to recommendations of the American Veterinary Medical Association Guide on Euthanasia (June 2007).

Coronary blood flow studies. Anesthetized dogs were intubated and ventilated with O2-supplemented air. Catheters were placed in the right femoral artery and vein as well as the left femoral artery. Blood pressure was measured from the right femoral artery catheter advanced into the aorta (and mean pressure and heart rate were derived from the pressure wave). Further, arterial blood was drawn from this catheter to analyze blood gases every 15–20 min; ventilatory adjustments were made as required to maintain normal parameters. Bicarbonate and supplemental anesthesia were given intravenously as needed. A left thoracotomy was performed at the fifth intercostal space. The left lung was restrained in gauze, and the pericardium was opened. A proximal portion of the left anterior descending coronary artery was dissected free of the myocardium in preparation for cannulation. Heparin (500 U/kg iv) was injected, and the extracorporeal perfusion system was primed with arterial blood from the left femoral. A ligature was tied securely around the proximal coronary artery, and the perfusion cannula was inserted distally. Perfusion pressure was adjusted to 100 mmHg and maintained with a servo-controlled roller pump drawing blood from the left femoral artery. Blood flow in the coronary perfusion line was measured with an in-line Transonic flow transducer. Hemodynamic variables were allowed to stabilize before experiments. For reactive hyperemia experiments, the roller pump was turned off and the coronary perfusion line was clamped for 30 s. 4-Aminopyridine (4-AP; 1 M) was dissolved in acidified water to enhance solubility and neutralize pH. This stock was diluted into saline and continuously infused into the coronary artery perfusion line by a syringe pump to reach a calculated coronary plasma concentration of 0.3 mM. The reactive hyperemia protocol was repeated during 4-AP infusion. Drugs were infused directly into the coronary circulation or given intravenously as indicated. With intracoronary infusion, the drug concentration was calculated for flow under basal conditions. 8-Phenyltheophylline (8-PT) was dissolved in equal parts of 1 N NaOH, ethanol, and propylene glycol and warmed. This stock solution of 8-PT was diluted into saline and infused to reach a calculated plasma concentration of 0.3 mM or injected intravenously at 5 mg/kg. L-NAME was dissolved in saline and infused at 1 mg/min ic. To determine the effect of 4-AP on vasodilation to adenosine and sodium nitroprusside (SNP), all drugs were given ic.

Isolated arterioles. For in vitro studies, hearts were collected from untreated dogs euthanized with sodium pentobarbital (100 mg/kg iv). An apical portion of the left ventricle was removed, and arterioles (~150 µm) were located under a dissecting microscope. Microvessels were dissected in a salt solution containing (mM) 145 NaCl, 4.7 KCl, 2 CaCl2, 1.17 MgSO4, 1.2 NaH2PO4, 5 glucose, 2 pyruvate, 0.02 EDTA, and 3 MOPS with 1% bovine serum albumin. This solution was buffered to pH 7.4 at 4°C or 37°C depending on whether it was used for dissection or experiments. Side branches were tied off if necessary, and arterioles were secured to glass micropipettes with 11-0 suture; this preparation was transferred to the stage of an inverted microscope. Experiments in arterioles with any flow at 60 mmHg were abandoned; diameter was measured by video edge detection.

Patch clamp. A 1- to 2-cm segment of left anterior descending coronary artery was cleaned and opened along its longitudinal axis. This ribbon of conduit artery was pinned lumen side up in a Sylgard-lined jar filled with physiological salt solution containing 0.5 mM Ca2+. The artery was covered with the same low-Ca2+ solution containing (mg/ml) 2 collagenase, 1 elastase, 1 soybean trypsin inhibitor, and 1 bovine serum albumin. The jar was placed in a shaking water bath at 37°C. After 30 min of enzymatic digestion, the enzyme solution was replaced with fresh low-Ca2+ solution. This was pipetted gently over the surface of the artery to liberate single cells. A suspension of cells was collected and kept at 4°C. Patch-clamp experiments were performed on freshly isolated myocytes within the next 8 h. Drops of cell suspension were added to a recording chamber mounted on an inverted microscope. Cells were continuously superfused with a salt solution containing (mM) 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, and 5 Tris, pH 7.4. Patch-clamp electrodes were fabricated from borosilicate glass and heat polished and had tip resistances of 2–4 M{Omega} when filled with solution containing (mM) 140 KCl, 1 MgCl2, 3 Mg-ATP, 1 Na-GTP, 0.1 EGTA, 10 HEPES, and 5 Tris, pH 7.1. After whole-cell access was established, series resistance and membrane capacitance were compensated as completely as possible. Currents were low-pass filtered at 1 kHz and digitized at 5 kHz. No voltage adjustments were made for a liquid junction potential calculated to be –4 mV.

Expression of KV1 mRNA. Total RNA was isolated from left anterior descending coronary arteries (SV Total RNA Isolation System, Promega), and mRNA was reverse transcribed to cDNA (ImProm-II Reverse Transcriptase System, Promega). Real-time PCR was performed to determine expression of KV1 family members (SYBR Green I, Molecular Probes; Taq DNA polymerase, Promega). Primers were designed with Genomatix DiAlign software and GenBank sequence information. Melt curves indicated a single PCR product in each reaction, and amplicons were sequenced to confirm identity. The expression of individual KV1 isoforms was normalized by using the threshold cycle (CT) number for each gene of interest, the average CT for all isoforms, and the equation 2Formula. Primer sequences are given in Table 1.


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  		Table 1. PCR primers

 
Statistics. In vivo and in vitro vasodilation data are expressed as means ± SE from n dogs. Hyperemic volume was calculated as area under the curve with Prism software (GraphPad, San Diego, CA). Perfusion territory was estimated with the methods established by Feigl and coworkers (20). Patch-clamp data are expressed as means ± SE from n cells. PCR experiments were performed in duplicate, and results are expressed as means ± SE from n dogs. Statistical comparisons were made with paired t-tests and one- or two-way repeated-measures analysis of variance (ANOVA) as appropriate. In all statistical tests, P < 0.05 was considered statistically significant. When overall treatment differences were indicated by ANOVA, a Bonferroni posttest was used to identify where differences existed.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
KV channels regulate resting coronary blood flow. KV channels control coronary blood flow at rest, and inhibiting them produces electrophysiological signs typically associated with myocardial ischemia (Fig. 1; n = 4). Coronary perfusion pressure was held at 100 mmHg, coronary vascular resistance was free to change, and flow was that necessary to maintain the pressure set point measured. 4-AP, an inhibitor of KV channels, was infused into the coronary artery to reach various calculated plasma concentrations. 4-AP reduced coronary flow in a concentration-dependent manner, with a significant effect observed at 0.1 mM (Fig. 1A). We simultaneously recorded the ECG in a modified lead II configuration. Prominent ST segment depression, suggesting possible myocardial ischemia, was noted in three of four dogs at 3 mM 4-AP (blood flow was reduced to 47 ± 8% of control; Fig. 1B). Infusions of 4-AP did not alter systemic hemodynamics (Table 2). We used 0.3 mM 4-AP for all experiments detailed below, because it was effective but did not alter the ECG.


Figure 1
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  		Fig. 1. Voltage-dependent K+ (KV) channels regulate resting coronary blood flow. Coronary blood flow (A) and ECG (B) were measured in open-chest anesthetized dogs (n = 4). 4-Aminopyridine (4-AP), at calculated plasma concentrations ([4-AP]), reduced coronary blood flow in a concentration-dependent manner. Asterisks in A indicate a significant reduction in coronary blood flow compared with control as determined by 1-way repeated-measures ANOVA and Bonferroni post hoc analysis. Asterisks in B indicate ST segment depression elicited by 3 mM 4-AP. ST segment depression with 4-AP was observed in 1 of 4 dogs at 1 mM, 3 of 4 dogs at 3 mM, and 4 of 4 dogs at 10 mM.

 

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  		Table 2. Hemodynamic variables before and after drugs

 
KV channels contribute to coronary reactive hyperemia. Reactive hyperemia in response to a 30-s interruption in coronary blood flow was measured in five dogs before and during intracoronary infusion of 4-AP (Fig. 2; Table 2). Baseline blood flow was 0.52 ± 0.08 ml·min–1·g–1. The peak hyperemic response was 3.00 ± 0.31 ml·min–1·g–1, and total hyperemic volume was 1.70 ± 0.16 ml/g. 4-AP reduced coronary blood flow 34 ± 5% (P < 0.05). Peak hyperemia was unaffected (94 ± 2% of control), but 4-AP reduced total hyperemic volume 32 ± 5% (P < 0.05). Classically, effects of inhibitors on reactive hyperemia have been assessed through debt repayment ratios; however, the debt repayment ratio was unchanged by 4-AP (7.4 ± 1.6 vs. 7.5 ± 1.6). This may be because 4-AP reduces baseline flow; therefore, we performed a series of experiments to match flow debt in the presence of 4-AP (i.e., increased the duration of ischemia; Fig. 3A). When flow debt in the presence of 4-AP was matched to 101 ± 7% (n = 4) of control by prolonging the duration of ischemia, the debt repayment ratio was reduced (Fig. 3B). Numerous studies indicate a role for glibenclamide-sensitive K+ channels in reactive hyperemia. To determine whether 4-AP has nonspecific effects on KATP channels, we tested whether 4-AP had any effect on vasodilation to pinacidil, a KATP channel opener. No inhibitory effect of 4-AP was observed (Fig. 4; n = 3).


Figure 2
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  		Fig. 2. KV channels contribute to vasodilation following a brief coronary artery occlusion. A: reactive hyperemia before and during intracoronary infusion of 0.3 mM 4-AP was measured in 5 dogs; a representative trace is shown. The left anterior descending coronary artery was occluded for 30 s and then released. Under control conditions, a large reactive hyperemic response was observed. 4-AP decreased baseline coronary blood flow and attenuated the hyperemic response. B: 4-AP-sensitive component of flow is shown. C–E: group data for effects of 4-AP on baseline flow (C), peak flow (D), and total hyperemic volume (E). *P < 0.05 by paired t-test.

 

Figure 3
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  		Fig. 3. 4-AP decreases the debt repayment ratio when flow debt is matched to the control condition. A: representative reactive hyperemia traces from a single dog. Blood flow responses were measured 1) under control conditions with a 30-s occlusion, 2) with 0.3 mM 4-AP and a 30-s occlusion, and 3) with 4-AP plus a 55-s occlusion. Flow debts were 12.9, 7.1, and 12.8 ml, respectively; thus ischemic debt with 4-AP was matched to the control level by prolonging the duration of occlusion. Matching the flow debt did not normalize the hyperemic response. B: group data (n = 4) demonstrating a 4-AP-induced reduction in the debt repayment ratio when flow debt is matched. *P < 0.05 by 1-way repeated-measures ANOVA and Bonferroni post hoc analysis.

 

Figure 4
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  		Fig. 4. 4-AP does not inhibit coronary vasodilation mediated by ATP-dependent K+ (KATP) channels. A: group data (n = 3) showing coronary blood flow responses to pinacidil before and during intracoronary infusion of 0.3 mM 4-AP. *P < 0.05 at baseline by 2-way repeated-measures ANOVA and Bonferroni post hoc analysis. B: pinacidil-induced changes in coronary blood flow before and during 4-AP infusion.

 
Comparative effects of 8-PT, L-NAME, and 4-AP on reactive hyperemia. Reactive hyperemia was measured in 13 dogs divided into two groups. In group 1 (n = 8), responses were measured under control conditions, during treatment with 8-PT (0.3 mM ic in 3 dogs or 5 mg/kg iv in 5 dogs), and with combined 8-PT and 4-AP. This experiment compared the contributions of adenosine and KV channels to reactive hyperemia. 8-PT virtually abolished vasodilation to adenosine (1 µg/kg ic), because increases in flow were only 12 ± 5% of the control response (P < 0.05). In group 2 (n = 5), responses were measured under control conditions, during intracoronary infusion of L-NAME (1 mg/min), and during combined treatment with L-NAME and 4-AP. This experiment compared the contributions of NO and KV channels to reactive hyperemia. L-NAME significantly inhibited vasodilation to acetylcholine (10 µg ic), because increases in flow were 36 ± 3% of control (P < 0.05).

8-PT had no effect on resting coronary flow (97 ± 4% of control) or peak hyperemia (98 ± 1% of control). Total hyperemic volume with 8-PT was 94 ± 5% of control (Fig. 5A), and only a small transient portion of reactive hyperemia was affected (Fig. 5B). 4-AP, in addition to 8-PT, reduced resting flow to 68 ± 4% of control (P < 0.05; Fig. 5C). Peak hyperemia with combined blockade was 96 ± 2% of control (Fig. 5D). Total hyperemic volume in the presence of 8-PT and 4-AP was 59 ± 6% of control (P < 0.05; Fig. 5E). L-NAME attenuated reactive hyperemia, and the combination of L-NAME and 4-AP further inhibited this response (Fig. 6, A and B). L-NAME reduced resting coronary blood flow 19 ± 7% (P < 0.05; Fig. 6C). In the presence of L-NAME, peak hyperemia was 102 ± 2% of control (Fig. 6D). Total hyperemic volume with L-NAME was reduced to 78 ± 4% of control (P < 0.05; Fig. 6E). 4-AP, in addition to L-NAME, reduced coronary flow to 54 ± 7% of control (P < 0.05; Fig. 6C). Peak hyperemia with L-NAME and 4-AP was 88 ± 5% of control (Fig. 6D), and total hyperemic volume was 52 ± 6% of control (P < 0.05; Fig. 6E).


Figure 5
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  		Fig. 5. Effects of 8-PT and 4-AP on reactive hyperemia. A: coronary blood flow traces from a representative dog. Reactive hyperemia was measured under control conditions, after treatment with 8-PT, and with 8-PT + 4-AP. B: flow inhibited by 8-PT or 8-PT + 4-AP, i.e., the 8-PT- and 4-AP-sensitive components of reactive hyperemia. C: group data (n = 8) for effect of 8-PT or 8-PT + 4-AP on baseline coronary blood flow. D: no effect of 8-PT ± 4-AP on peak flow. E: effect of 8-PT ± 4-AP on total hyperemic volume. *P < 0.05 by 1-way repeated-measures ANOVA and Bonferroni post hoc analysis.

 

Figure 6
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  		Fig. 6. Effects of NG-nitro-L-arginine methyl ester (L-NAME) and 4-AP on reactive hyperemia. A: coronary blood flow traces from a representative dog. Reactive hyperemia was measured under control conditions, during intracoronary infusion of L-NAME (1 mg/min), and with L-NAME + 4-AP (coronary plasma concentration = 0.3 mM). B: flow inhibited by L-NAME or L-NAME + 4-AP, i.e., differences between control and L-NAME as well as L-NAME and L-NAME + 4-AP. C: group data (n = 5) for inhibitory effect of L-NAME ± 4-AP on baseline coronary blood flow. D: no effect of L-NAME ± 4-AP on peak flow. E: inhibitory effect of L-NAME and 4-AP on total hyperemic volume. *P < 0.05 by 1-way repeated-measures ANOVA and Bonferroni post hoc analysis.

 
Adenosine-induced coronary vasodilation involves KV channels. Vasodilation in response to graded intracoronary doses of adenosine was determined in five dogs before and during intracoronary infusion of 4-AP. Adenosine increased blood flow in a dose-dependent manner (Fig. 7A). Blood flow was allowed to return to baseline after the adenosine dose-response protocol, and 4-AP was administered. 4-AP reduced blood flow 26 ± 3% (P < 0.05) and attenuated adenosine-induced coronary blood flow responses (Fig. 7A; Table 2). Reduced responses in the presence of 4-AP were not due to tachyphylaxis from repeated adenosine administration, because ceasing the 4-AP infusion allowed baseline blood flow to normalize (107 ± 5% of control) and vasodilation to return to normal with a third adenosine infusion (105 ± 8% of control at 9 µg/min).


Figure 7
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  		Fig. 7. KV channels function in adenosine-induced coronary vasodilation. A: adenosine-induced vasodilation was measured before and during intracoronary infusion of 4-AP (0.3 mM plasma concentration; n = 5). 4-AP decreased baseline coronary blood flow (indicated by dashed line) and attenuated responses to adenosine. B: adenosine-induced dilation of isolated coronary arterioles before and after treatment with 0.3 mM 4-AP (n = 5). C: adenosine-induced dilation of isolated coronary arterioles before and after treatment with 1 µM correolide, a selective inhibitor of KV1 channels (n = 4). Data were compared by 2-way repeated-measures ANOVA; *P < 0.05 by Bonferroni post hoc test.

 
KV1 channels in adenosine-induced arteriolar dilation. Coronary arterioles were cannulated and pressurized to 60 mmHg for video analysis of diameter. Endothelin-1 (1 nM) was used to increase active tone before adenosine concentration-response protocols. Dilator responses to adenosine were determined before and after treatment with 4-AP or correolide. Correolide selectively inhibits KV channels containing KV1 subunits (23). Adenosine dilated arterioles in a concentration-dependent manner, and this was inhibited by 4-AP (Fig. 7B; n = 5). Diameter with active tone was 81 ± 16 and 77 ± 10 µm in the absence and presence of 4-AP, respectively. Maximum passive diameter was 144 ± 15 µm with 100 µM SNP (an NO donor) and 100 µM verapamil (an L-type Ca2+ channel blocker). Correolide (1 µM) inhibited adenosine-induced dilation (Fig. 7C; n = 4). Diameter with active tone was 60 ± 6 and 45 ± 8 µm before and after correolide, respectively. Maximum passive diameter was 150 ± 18 µm.

KV channels in NO-induced vasodilation. Experiments similar to the adenosine experiments described above were conducted with SNP, which increased blood flow in a dose-dependent manner (Fig. 8A). The SNP dose-response protocol was repeated with 4-AP treatment. 4-AP reduced resting flow 19 ± 3% (P < 0.05) and attenuated SNP-induced coronary blood flow responses (Fig. 8A; Table 2). Dilator responses of arterioles to SNP were determined before and after treatment with 1 µM correolide. Under control conditions, SNP dilated arterioles in a concentration-dependent manner (Fig. 8B; n = 5). Correolide inhibited SNP-induced dilation (Fig. 8B). Diameter with active tone was 45 ± 15 and 44 ± 7 µm before and after correolide, respectively. Maximum passive diameter was 143 ± 9 µm.


Figure 8
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  		Fig. 8. KV channels participate in nitric oxide-induced vasodilation. A: vasodilation to sodium nitroprusside (SNP) was measured before and during intracoronary infusion of 4-AP (0.3 mM plasma concentration; n = 3). 4-AP decreased baseline coronary blood flow (indicated by dotted line) and attenuated responses to SNP. B: SNP-induced dilation of isolated coronary arterioles before and after treatment with 1 µM correolide, a selective inhibitor of KV1 channels (n = 5). Data were compared by 2-way repeated-measures ANOVA; *P < 0.05 by Bonferroni post hoc test.

 
Correolide-sensitive KV current. Whole cell patch-clamp techniques were used to measure K+ current in coronary vascular smooth muscle cells and determine the sensitivity of KV current to correolide (Fig. 9). K+ currents were measured with solutions designed to mimic physiological ion concentrations. The intracellular and extracellular concentrations of K+ were 140 and 5 mM, respectively. The bath solution contained 2 mM Ca2+; the pipette solution contained 0.1 mM EGTA and no added Ca2+. The pipette solution also contained 3 mM ATP. Voltage-clamping cells to voltages between –100 and +100 mV elicited time- and voltage-dependent current. We demonstrated previously (43) that current under these conditions is mediated largely by KV and BKCa channels. Current at –80 mV, the holding potential, was –20 ± 5 pA, and the whole cell current reversed direction at –41 ± 5 mV (n = 6). Correolide significantly inhibited current (Fig. 9, B and E). The current blocked by correolide was voltage dependent, increasing at approximately –30 mV (Fig. 9E, inset). Conductance, i.e., current divided by driving force, saturated at +50 mV.


Figure 9
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  		Fig. 9. Correolide-sensitive KV1 current in coronary smooth muscle cells. Representative patch-clamp recordings from a coronary vascular smooth muscle cell before (A) and after (B) treatment with 1 µM correolide are shown. The correolide-sensitive component of current, i.e., A minus B, is shown in C. D: voltage template used to elicit current. E: group data (n = 6) for inhibitory effect of correolide on K+ current. Inset, correolide-sensitive current. *P < 0.05 by 2-way repeated-measures ANOVA and Bonferroni post hoc analysis.

 
Expression of KV1 mRNA in canine coronary artery. Delayed rectifier current in conduit coronary artery smooth muscle was inhibited by correolide, indicating that functional channels contain subunits of the KV1 family. We used reverse transcriptase PCR techniques to screen conduit coronary arteries for expression of mRNA encoding pore-forming {alpha}-subunits of KV1 channels (Fig. 10). There are eight members of the KV1 family (KV1.1 through KV1.8). KV1.4 was not assayed, because its expression is associated with transient A-type currents, not delayed rectifier currents (22). Expression of KV1.1, KV1.2, KV1.3, KV1.5, KV1.6, KV1.7, and KV1.8 transcripts was observed. Total RNA was isolated from conduit coronary arteries of four different dogs, and experiments were performed in duplicate. When the relative expression of individual KV1 channel isoforms was compared, no significant differences were observed.


Figure 10
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  		Fig. 10. KV1 subunit expression in the canine coronary artery. Quantitative real-time PCR results for the expression of KV1 {alpha}-subunits relative to each other. Assays were run in duplicate from 4 dogs. ND = not determined, because cloned KV1.4 mediates an A-type K+ current rather than a delayed rectifier K+ current. Abundance of any particular KV channel isoform is expressed relative to the others.

 
Biophysical properties of KV current. Cloned KV1 channels have characteristic properties that can aid in the molecular identification of native delayed rectifier channels; these properties include the voltage of half activation (V1/2), the slope factor of voltage-dependent activation (Ka), and sensitivity to block by 4-AP (EC50). We determined the V1/2, Ka, and 4-AP EC50 of the delayed rectifier current in smooth muscle cells from conduit coronary arteries (Fig. 11). Tail currents at –40 mV were measured after stepping the cells from –100 to +100 mV (see voltage protocol in Fig. 9D). Tails were normalized, plotted vs. voltage, and fit with a Boltzmann function. V1/2 was –12.3 ± 1.2 mV, and Ka was 7.1 ± 0.4 (n = 17; Fig. 11A). Current at 0 mV was blocked by 4-AP with an EC50 of 1.1 ± 0.1 mM (n = 6; Fig. 11B).


Figure 11
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  		Fig. 11. Biophysical and pharmacological characteristics of KV current in canine coronary smooth muscle cells. A: activation curve from which the voltage of half activation (V1/2) and slope factor (Ka) were determined (n = 17). The voltage template shown in Fig. 9D was used. Tail currents were measured at –40 mV, normalized, plotted vs. voltage, and fit with a Boltzmann function. G, conductance. B: 4-AP sensitivity of KV current at 0 mV (n = 6).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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We tested the hypothesis that KV channels participate in coronary reactive hyperemia. Further, we examined the role of KV channels in responses to two potential mediators of reactive hyperemia, NO and adenosine. Major new findings are that 1) KV channels regulate resting coronary blood flow, 2) 4-AP shapes reactive hyperemia by altering its duration (but has no effect on its peak), and 3) KV1 channels participate in adenosine- and NO-induced coronary vasodilation. Our data support previous observations that KV channels participate in adenosine-induced coronary vasodilation in vitro (21, 24, 25). We extend those findings with in vivo studies and demonstrate that adenosine-activated channels are correolide sensitive, and thus contain subunits of the KV1 family. Similarly, our data support previous studies showing that 4-AP-sensitive KV channels mediate NO-induced vasodilation (33, 34, 49, 50, 59). We enhance the understanding of KV channels regulated by NO by demonstrating that they contain correolide-sensitive KV1 subunits. These findings, together with our previous studies of coronary vascular KV channels (43, 44, 47), suggest that KV1 channels serve as end-effectors that integrate multiple physiological, pathophysiological, and pharmacological stimuli to regulate coronary microvascular resistance.

We demonstrate that KV channels contribute significantly to the regulation of coronary vascular resistance under basal or resting conditions. 4-AP decreased coronary blood flow in a concentration-dependent manner, and this was associated with marked ST segment depression. We did not measure coronary sinus PO2 or myocardial lactate production; therefore, we can only speculate that the observed ST segment depression is the result of myocardial ischemia. Consistent with the idea that 4-AP-induced ST segment depression is secondary to ischemia, we have observed reversal of this phenomenon with vasodilators (e.g., bradykinin) in other experiments. It is unlikely that 4-AP directly affects the ST segment of the ECG for the following reasons. First, 4-AP does not alter the resting membrane potential of cardiac muscle (31); therefore, depolarization of the drug-perfused region is unlikely. Second, if a drug were to transmurally depolarize the perfused region, this would elevate, not depress, the ST segment of lead II. Thus 4-AP-induced ST segment depression most likely results from subendocardial ischemia caused by the marked decrease in coronary blood flow. The contribution of 4-AP-sensitive KV channels to baseline coronary vascular resistance appears to be greater than that of other K+ channels investigated previously. Specifically, earlier studies found a ~10–25% reduction in coronary blood flow following blockade of KATP channels with glibenclamide (13, 15, 19, 42) and little or no change in coronary flow following inhibition of BKCa channels with iberiotoxin or charybdotoxin (30, 36, 40). We interpret these findings to indicate that vasodilator substances released by cardiac myocytes and endothelium under basal conditions regulate coronary microvascular resistance via pathways that converge largely upon KV channels.

The phenomenon and mechanisms of coronary reactive hyperemia have been studied intensively for decades, and two candidate mediators, adenosine and NO, have received the most support. Building on observations of Katz and Lindner (29) in fibrillating hearts, Coffman and Gregg (10, 11) pioneered study of coronary reactive hyperemia in the working heart. They observed that hyperemic flow increased with the duration of occlusion and postulated a similar increase in the factor(s) responsible. Berne (7) suggested adenosine as mediator of hypoxic vasodilation, and others extended this to reactive hyperemia. For example, Olsson and colleagues (39) performed a kinetic analysis of adenosine washout during reactive hyperemia and suggested that adenosine levels account for coronary flow changes. Additionally, exogenous adenosine (at levels comparable to ischemic production) elicits vasodilation equal to that observed in reactive hyperemia (38). Finally, adenosine deaminase reversibly reduces hyperemic flow (without any effect on peak) following an occlusion longer than 5 s (46). These data solidify the place of adenosine on the list of viable mediators of reactive hyperemia. Importantly, however, other studies provide compelling data to indicate that adenosine plays no role in coronary reactive hyperemia (8). Our data are more like those of Bittar and Pauly (8) than those of Saito et al. (46). The development of arginine analogs soon placed NO on the list of mediators of reactive hyperemia. Specifically, Yamabe et al. (56) demonstrated that blocking NO production reduced hyperemic volume, without any effect on peak hyperemia. This was confirmed by Smith and Canty (48), who showed that NO modulated autoregulatory responses and functioned in reactive hyperemia, including peak hyperemic flow. Similarly, Altman et al. (2) demonstrated that NO contributed to hyperemic flow (without effect on peak) but, importantly, had no major role in metabolic vasodilation.

The end-effectors (i.e., smooth muscle targets) producing responses to adenosine, NO, and other mediators in reactive hyperemia are not entirely understood, but our present work and the studies of others indicate that KATP and KV channels are involved (2, 6, 21, 24–26, 33, 34, 49, 50, 59). Earlier studies suggested that a significant portion of reactive hyperemia is mediated by KATP channels (9, 15) and showed that subsequent inhibition of adenosine receptors and NO synthesis essentially abolishes the response (15, 26). Glibenclamide, the KATP channel antagonist used, significantly attenuates coronary vasodilation to adenosine (6, 9, 15, 26), may block KV channels (57), but does not affect coronary responses to the NO donor SNP (15, 26). Thus relative contributions of KATP and KV channels to the responses were unable to be defined. We demonstrate that inhibition of KV channels with 4-AP significantly reduces hyperemic flow, without affecting coronary vasodilation to the KATP channel agonist pinacidil, indicating that KV channels contribute to reactive hyperemia by shaping the duration of the response.

We show that 4-AP significantly reduces total hyperemic volume, suggesting that endogenous vasodilators activate KV channels to produce coronary vasodilation. Adenosine and NO are the candidate mediators of coronary reactive hyperemia that have received the most experimental support. We show that coronary vasodilation in response to adenosine and NO is mediated, at least in part, via KV1 channels. We demonstrated the efficacy of 8-PT to inhibit adenosine-induced vasodilation; however, our reactive hyperemia experiments with 8-PT indicated only a small role for adenosine in coronary reactive hyperemia. This finding is similar to that of Bittar and Pauly (8) but is somewhat at odds with previous studies (46). The reasons for this discrepancy are unclear. Regardless, we show that a significant portion of adenosine-induced dilation is mediated via KV1 channels. Thus, even if our studies have completely underestimated the role of adenosine in reactive hyperemia, our data do indicate a definitive role for KV channels in adenosine-induced dilation. Our studies indicate a clear role for NO in coronary reactive hyperemia; this is entirely consistent with earlier studies. We extend those studies by demonstrating that KV1 channels serve as targets for NO-induced vasodilation. It is important to note that 4-AP further reduced reactive hyperemia in the presence of L-NAME or 8-PT, indicating that mediators other than NO and adenosine contribute to ischemic vasodilation by activating KV channels. One potential, but untested, mediator of reactive hyperemia is H2O2, which we previously demonstrated (43, 44, 47) mediates coronary metabolic vasodilation via KV channels. The role of H2O2 in metabolic vasodilation has recently been confirmed by others (32, 55), but the hypothesis that H2O2 functions in reactive hyperemia remains to be addressed in future studies.

The question arises as to whether the effect of 4-AP on reactive hyperemia is one that would be expected from "simple" vasoconstriction, as 4-AP reduced baseline coronary blood flow by increasing coronary vascular resistance. The experiments in Fig. 3 (matched flow debts) and previous reports suggest this is an unlikely explanation, because studies indicate that reactive hyperemia is unaffected by exogenous vasoconstrictors. For example, infusing endothelin into the coronary circulation of pigs had no effect on peak reactive hyperemia even when coronary blood flow was reduced severely enough to cause lactate production (53). Similarly, studies of reactive hyperemia in the human forearm show that vasoconstrictor doses of norepinephrine had no major effect; baseline flow was reduced, but minimal postischemic vascular resistance was unchanged (41). Curiously, nifedipine and diltiazem, inhibitors of L-type Ca2+ channels, have been reported to decrease coronary reactive hyperemia without altering resting blood flow (16). These observations suggest that there is no relationship between preischemic arteriolar tone and the peak of reactive hyperemia; therefore, we suggest that the inhibitory effect of 4-AP on reactive hyperemia is specifically related to block of endogenous vasodilator pathways that regulate the duration of hyperemic flow. This conclusion is similar to those derived from studies using inhibitors of KATP channels, nitric oxide synthase, and adenosine receptors.

There are several characteristics of reactive hyperemia to analyze and interpret. Traditionally, the major features examined are hyperemic volume and the repayment of flow debt (37). Peak hyperemic flow is invariably reported but rarely discussed or interpreted. Peak flow increases with the duration of ischemia for occlusions up to 15–30 s; longer occlusions do not further reduce the minimum vascular resistance, suggesting that the absolute vasodilator capacity has been reached with 30 s of ischemia. Accordingly, peak hyperemic flow to a maximal ischemic stimulus has become thought of more as a morphological index (i.e., the integrated arteriolar lumen) rather than a functional end point. However, because peak flow is not altered by 4-AP, one could conclude that KV channels do not contribute to ischemic vasodilation (Ref. 47; i.e., block of KV channels does not affect minimum vascular resistance). Importantly, however, 4-AP dramatically shortens the duration of reactive hyperemia and reduces total hyperemic volume. This point deserves emphasis when one considers that the seemingly excessive repayment of flow debt provides only ~80% oxygen repayment (45). Thus it seems logical that any intervention that decreases hyperemic volume would further impinge upon this already meager oxygen repayment. Our data indicate that KV channels determine total hyperemic volume, an important functional aspect of coronary reactive hyperemia.

Previously, we demonstrated (43, 44, 47) that 4-AP-sensitive KV channels function as targets for vasodilation in response to myocardial metabolism and H2O2. Here we further investigated the molecular, biophysical, and pharmacological properties of KV current in smooth muscle cells from conduit coronary arteries. A large proportion of KV current was correolide sensitive; therefore, this rules out a major contribution of other KV channel families, unless they are formed into heteromultimers with KV1 family members. The V1/2, Ka, and 4-AP EC50 of the native delayed rectifier, compared with properties of cloned KV channels (22), support the idea that this current is likely mediated by members of the KV1 family. The properties of the native current make KV1.5 a probable candidate; this isoform has been demonstrated to comprise a portion of the KV current in other smooth muscle cells (1, 51). Future studies will be directed at identifying the molecular composition of the delayed rectifier channel(s) in canine coronary smooth muscle. It will be important to study ion channels in smooth muscle cells from resistance arteries of the microcirculation, because these control coronary vascular flow. A limitation of this report is that the patch clamp and molecular studies were performed on smooth muscle cells from conduit arteries, which may not be relevant to the regulation of coronary vascular resistance.

In conclusion, data from this investigation support the hypothesis that KV1 channels serve as important end-effectors integrating multiple relevant stimuli to regulate coronary microvascular resistance. Our data show that KV channels regulate resting coronary blood flow and shape reactive hyperemia by altering duration. Further, our data show that KV1 channels participate in adenosine- and NO-induced coronary vasodilation. No drugs currently exist that are openers of KV channels; however, the advent of such agents might be as clinically significant as the development of KATP channel openers. Our data raise the interesting possibility that decreases in the function, expression, and/or signaling to KV channels could be an important mechanism contributing to impaired control of coronary blood flow in many disease states, including obesity, hypertension, and heart failure.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Our work was supported by National Heart, Lung, and Blood Institute Grant HL-67804.


   ACKNOWLEDGMENTS
 
We thank the laboratory of Dr. Susan J. Gunst for providing canine hearts for our in vitro studies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. D. Tune, Dept. of Cellular and Integrative Physiology, Indiana Univ. School of Medicine, 635 Barnhill Dr., Rm. MS 2063, Indianapolis, IN 46202-5120 (e-mail: jtune{at}iupui.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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